Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds.

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Methodology Sample preparation
Mouse SVF was isolated from inguinal white adipose tissue of adult BALB/C, mTmG, Cdh5-CreER/mTmG or Apln-CreER/ mTmG mice. Human SVF was isolated from subcutaneous lipoaspirate of thigh, abdomen or hip. The SVF of diabetic patients was isolated from the abdominal subcutaneous adipose tissue. The adipose tissue was rinsed with calcium-and bicarbonatefree Hank's solution with HEPES (CBFHH) and minced using fine scissors until the tissue suspension appeared homogeneous. The tissue was transferred in gentleMACS C Tubes (Miltenyi Biotec, 130-093-237, 130-096-334) containing a pre-warmed digestion solution composed of 1 mg/mL Collagenase NB4 Standard Grade (Nordmark Biochemicals, S1745401), 0.5 mg/mL DNase II (Sigma, D8764) and 100 μg/mL antibiotics (gentamicin, penicillin/streptomycin) dissolved in BFHH (Bicarbonate-Free Hanks´solution with HEPES) supplemented with 2mM CaCl2. GentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427) was used to digest the tissue running the program 37C_mr_ATDK_1. The digested tissue was then diluted in Dulbecco's Modified Eagle Medium (DMEM, Sigma) supplemented with 10% foetal bovine serum (FBS) and centrifuged at 700 x g for 10 minutes. After removing the oily and liquid layers, the pellet was resuspended in DMEM with 10% FBS and filtered using a 70 μm pore size cell strainer (Falcon, 352350). The cell suspension was centrifuged at 500 x g for 8 minutes and resuspended in FACS buffer with human Fc blocking antibody (dilution 1:100, BD Pharmigen, 564219). After 10 minutes, the cells were centrifuged at 500 x g for 5 minutes and resuspended in staining-cocktail for 25 minutes on ice, protected from light. After incubation, cells were rinsed twice with FACS buffer and fixed in 4% PFA for acquisition.

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Flow data were analyzed with Diva software (BD Bioscience) or FlowJo software (Tree Star, Inc.).

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The purity of sorted cells was determined by post-sort analysis through immunofluorescence analysis as shown in Supplementary Fig 12B. Gating strategy Gating strategies is described in Figure 4I and Supplementary Fig 1F and 12A. Gates are always based on initial debris and doublets exclusion based on physical parameters. Gates are drawn on FMO controls.
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